Methods and compositions for overcoming resistance to biologic and chemotherapy

ABSTRACT

This invention provides a method for identifying potential therapeutic agents by contacting a target cell with a candidate therapeutic agent which is a selective substrate for an endogenous, intracellular enzyme in the cell which is enhanced in its expression as a result of selection by biologic or chemotherapy. This invention also provides methods and examples of molecules for selectively killing a pathological cell by contacting the cell with prodrug that is a selective substrate for an endogenous, intracellular enzyme. The prodrug is subsequently converted to a cellular toxin. Further provided by this invention is a method for treating a pathology characterized by pathological, hyperproliferative cells in a subject by administering to the subject a prodrug that is a selective substrate for an endogenous, overexpressed, intracellular enzyme, and converted by the enzyme to a cellular toxin in the hyperproliferative cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. provisional application No.60/055,525, filed Aug. 8, 1997.

TECHNICAL FIELD

The present invention relates to the field of drug discovery andspecifically, the design of prodrugs which are substrates for anintracellular enzyme critical to resistance to cancer therapeutics inpathological cells and converted to a cell toxin by the intracellularenzyme.

BACKGROUND

Throughout this disclosure, various publications are referenced by firstauthor and date, patent number or publication number. The fullbibliographic citation for each reference can be found at the end ofthis application, immediately preceding the claims. The disclosures ofthese references are hereby incorporated by reference into thisdisclosure to more fully describe the state of the art to which thisinvention pertains.

Cancer cells are characterized by uncontrolled growth,de-differentiation and genetic instability. The instability expressesitself as aberrant chromosome number, chromosome deletions,rearrangements, loss or duplication beyond the normal dipoid number.Wilson, J. D. et al. (1991). This genomic instability may be caused byseveral factors. One of the best characterized is the enhanced genomicplasticity which occurs upon loss of tumor suppression gene function(e.g., Almasan A. et al. (1995)). The genomic plasticity lends itself toadaptability of tumor cells to their changing environment, and may allowfor the more frequent mutation, amplification of genes, and theformation of extrachromosomal elements (Smith, K. A. et al. (1995) andWilson, J. D. et al. (1991)). These characteristics provide formechanisms resulting in more aggressive malignancy because it allows thetumors to rapidly develop resistance to natural host defense mechanisms,biologic therapies (Wilson, J. D. et al. (1991) and Shepard, H. M. etal. (1988)), as well as to chemotherapeutics. Almasan, A. et al. (1995)and Wilson, J. D. et al. (1991).

Cancer is one of the most commonly fatal human diseases worldwide.Treatment with anticancer drugs is an option of steadily increasingimportance, especially for systematic malignancies or for metastaticcancers which have passed the state of surgical curability.Unfortunately, the subset of human cancer types that are amenable tocurative treatment today is still rather small (Haskell, C. M. eds.(1995), p. 32). Progress in the development of drugs that can cure humancancer is slow. The heterogeneity of malignant tumors with respect totheir genetics, biology and biochemistry as well as primary ortreatment-induced resistance to therapy mitigate against curativetreatment. Moreover, many anticancer drugs display only a low degree ofselectivity, causing often severe or even life threatening toxic sideeffects, thus preventing the application of doses high enough to killall cancer cells. Searching for anti-neoplastic agents with improvedselectivity to treatment-resistant pathological, malignant cells remainstherefore a central task for drug development. In addition, widespreadresistance to antibiotics is becoming an important, world-wide, healthissue. Segovia, M. (1994) and Snydman, D. R. et al. (1996).

Classes of Chemotherapeutic Agents

The major classes of agents include the alkylating agents, antitumorantibiotics, plant alkaloids, antimetabolites, hormonal agonists andantagonists, and a variety of miscellaneous agents. See Haskell, C. M.,ed., (1995) and Dorr, R. T. and Von Hoff, D. D., eds. (1994).

The classic alkylating agents are highly reactive compounds that havethe ability to substitute alkyl groups for the hydrogen atoms of certainorganic compounds. Alkylation of nucleic acids, primarily DNA, is thecritical cytotoxic action for most of these compounds. The damage theycause interferes with DNA replication and RNA transcription. The classicalkylating agents include mechlorethamine, chlorambucil, melphalan,cyclophosphamide, ifosfamide, thiotepa and busulfan. A number ofnonclassic alkylating agents also damage DNA and proteins, but throughdiverse and complex mechanisms, such as methylation or chloroethylation,that differ from the classic allylators. The nonclassic alkylatingagents include dacarbazine, carmustine, lomustine, cisplatin,carboplatin, procarbazine and altretamine.

The clinically useful antitumor drugs are natural products of variousstrains of the soil fungus Streptomyces. They produce their tumoricidaleffects by one or more mechanisms. All of the antibiotics are capable ofbinding DNA, usually by intercalation, with subsequent unwinding of thehelix. This distortion impairs the ability of the DNA to serve as atemplate for DNA synthesis, RNA synthesis, or both. These drugs may alsodamage DNA by the formation of free radicals and the chelation ofimportant metal ions. They may also act as inhibitors of topoisomeraseII, an enzyme critical to cell division. Drugs of this class includedoxorubicin (Adriamycin), daunorubicin, idarubicin, mitoxantrone,bleomycin, dactinomycin, mitomycin C, plicamycin and streptozocin.

Plants have provided some of the most useful antineoplastic agents.Three groups of agents from this class are the Vinca alkaloids(vincristine and vinblastine), the epipodophyllotoxins (etoposide andteniposide) and paclitaxel (Taxol). The Vinca alkaloids bind tomictotubular proteins found in dividing cells and the nervous system.This binding alters the dynamics of tubulin addition and loss at theends of mitotic spindles, resulting ultimately in mitotic arrest.Similar proteins make up an important part of nervous tissue; therefore,these agents are neurotoxic. The epipodophyllotoxins inhibittopoisomerase II and therefore have profound effects on cell function.Paclitaxel has complex effects on microtubules.

The antimetabolites are structural analogs of normal metabolites thatare required for cell function and replication. They typically work byinteracting with cellular enzymes. Among the many antimetabolites thathave been developed and clinically tested are methotrexate,5-fluorouracil (5-FU), floxuridine (FUDR), cytarubine, 6-mercaptopurine(6-MP), 6-thioguanine, deoxycoformycin, fludarabine,2-chlorodeoxyadenosine, and hydroxyurea.

Endocrine manipulation is an effective therapy for several forms ofneoplastic disease. A wide variety of hormones and hormone antagonistshave been developed for potential use in oncology. Examples of availablehormonal agents are diethylstilbestrol, tamoxifen, megestrol acetate,dexamethasone, prednisone, aminoglutethimide, leuprolide, goserelin,flutamide, and octreotide acetate.

Drawbacks of Current Chemotherapeutic Agents

Among the problems currently associated with the use of chemotherapeuticagents to treat cancers are the high doses of agent required; toxicitytoward normal cells, i.e., lack of selectivity; immunosuppression;second malignancies; and drug resistance.

The majority of the agents that are now used in cancer chemotherapy actby an anti-proliferative mechanism. However, most human solid cancers donot have a high proportion of cells that are rapidly proliferating andthey are therefore not particularly sensitive to this class of agent.Moreover, most antineoplastic agents have steep dose-response curves.Because of host toxicity, treatment has to be discontinued at doselevels that are well below the dose that would be required to kill allviable tumor cells.

Another side effect associated with present day therapies is the toxiceffect of the chemotherapeutic on the normal host tissues that are themost rapidly dividing, such as the bone marrow, gut mucosa and cells ofthe lymphoid system. The agents also exert a variety of other adverseeffects, including neurotoxicity; negative effects on sexuality andgonadal function; and cardiac, pulmonary, pancreatic and hepatictoxicities; vascular and hypersensitivity actions, and dermatologicalreactions.

Hematologic toxicity is the most dangerous form of toxicity for many ofthe antineoplastic drugs used in clinical practice. Its most common formis neutropenia, with an attendant high risk of infection, althoughthrombocytopenia and bleeding may also occur and be life threatening.Chemotherapy may also induce qualitative defects in the function of bothpolymorphonuclear leukocytes and platelets. The hematopoieic growthfactors have been developed to address these important side effects.Wilson, J. D. et al. (1991) and Dorr, R. T. and Von Hoff D. D., eds.(1994).

Most of the commonly used antineoplastic agents are capable ofsuppressing both cellular and humoral immunity. Infections commonly leadto the death of patients with advanced cancer, and impaired immunity maycontribute to such deaths. Chronic, delayed immunosuppression may alsoresult from cancer chemotherapy.

The major forms of neurotoxicity are arachnoiditis; myelopathy orencephalomyelopathy; chronic encephalopathies and the somnolencesyndrome; acute encephalopathies; peripheral neuropathies; and acutecerebellar syndromes or ataxia.

Many of the commonly employed antineoplastc agents are mutagenic as wellas teratogenic. Some, including procarbazine and the alkylating agents,are clearly carcinogenic. This carcinogenic potential is primarily seenas delayed acute leukemia in patients treated with polyfunctionalalkylating agents and inhibitors of topoisomerase II, such as etoposideand the anthracycline antibiotics. Chemotherapy has also been associatedwith cases of delayed non-Hodgkin's lymphoma and solid tumors. Thepresent invention will minimize these effects since the prodrug willonly be activated within tumor cells.

The clinical usefulness of a chemotherapeutic agent may be severelylimited by the emergence of malignant cells resistant to that drug. Anumber of cellular mechanisms are probably involved in drug resistance,e.g., altered metabolism of the drugs, impermeability of the cell to theactive compound or accelerated drug elimination from the cell, alteredspecificity of an inhibited enzyme, increased production of a targetmolecule, increased repair of cytotoxic lesions, or the bypassing of aninhibited reaction by alternative biochemical pathways. In some cases,resistance to one drug may confer resistance to other, biochemicallydistinct drugs. Amplification of certain genes is involved in resistanceto biologic and chemotherapy. Amplification of the gene encodingdihydrofolate reductase is related to resistance to methotrexate, whileamplification of the gene encoding thymidylate synthase is related toresistance to treatment with 5-fluoropyrimidines. Table 1 summarizessome prominent enzymes in resistance to biologic and chemotherapy. TABLE1 Enzymes Overexpressed in Resistance to Chemotherapy Biologic or EnzymeChemotherapy Referenced (Examples) Thymidylate Uracil-based Lönn, U. etal. synthase Folate-based Kobayashi, H. et al. Quinazoline-basedJackman, AL et al. Dihydrofolate Folate-based Banerjee, D. et al.reductase Bertino, J. R. et al. Tyrosine kinases TNF-alpha Hudziak, R.M. et al. Multidrug Stuhlinger, M. et al. resistance MDR-associatedMultidrug Simon, S. M. and Schindler, proteins (ABC P-gp resistance M.Gottesman, M. M. et al. proteins) CAD* PALLA** Smith, K. A. et al. Dorr,R. T. and Von Hoff, D. D., eds. Ribonucleotide Hydroxyurea Wettergren,Y. et al. reductase Yen, Y. et al.*CAD = carbamyl-P synthase, aspartate transcarbamylase, dihydrocrotase**PALA = N-(phosphonacetyl)-L-aspartateUse of Prodrugs as a Solution to Enhance Selectivity of a ChemotherapyAgent

The poor selectivity of anticancer agents has been recognized for a longtime and attempts to improve selectivity and allow greater doses to beadministered have been numerous. One approach has been the developmentof prodrugs. Prodrugs are compounds that are toxicologically inert butwhich may be converted in vivo to active toxic products. In some cases,the activation occurs through the action of a non-endogenous enzymesdelivered to the target cell by antibody (“ADEPT” or antibody-dependentenzyme prodrug therapy (U.S. Pat. No. 4,975,278)) or gene targeting(“GDEPT” or gene dependent enzyme-prodrug therapy (Melton, R. G. andSherwood, R. F. (1996)). These technologies have severe limitations withrespect to their ability to exit the blood and penetrate tumors.Connors, T. A. and Knox, R. J. (1995).

Accordingly, there is a need for more selective agents which canpenetrate the tumor and inhibit the proliferation and/or kill cancercells that have developed resistance to therapy. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

This invention provides a method for identifying therapeutic agents bycontacting a target or test cell with a candidate therapeutic agent orprodrug which is a selective substrate for a target enzyme in the cell.In one embodiment, the target enzyme is an endogenous, intracellularenzyme which is overexposed and confers resistance to biologic andchemotherapeutic agents. In a separate embodiment, the activity of theenzyme has been greatly enhanced in a tumor cell as a result of loss oftumor suppressor function (Smith, K. A. et al. (1995) and Li, W. et al.(1995)) and/or selection resulting from previous exposure tochemotherapy, (Melton, R. G. and Sherwood, R. F. (1996)). In a separateembodiment, the target enzyme is an expression product of a foreign genein the cell, wherein the foreign gene encodes a target enzyme.

After the cell is contacted in vitro and/or in vivo with the candidateprodrug, the cell is assayed for efficacy of the agent by noting if theagent caused a reduction in cellular proliferation or if the agent killsthe cell. In one aspect of this invention, the prodrug kills the cell orinhibits the cellular proliferation by the release of a toxic byproductfrom the prodrug by the target enzyme. In a further aspect of thisinvention, one or more “target enzymes” can be used to activate theprodrug so that it releases the toxic byproduct.

Another aspect of this invention includes kits for use in assaying fornew prodrugs having the characteristics described herein against targetenzymes. The kits comprise the reagents and instructions necessary tocomplete the assay and analyze the results.

This invention also provides methods and examples of molecules forselectively killing a pathological cell by contacting the cell with aprodrug that is a selective substrate for a target enzyme, e.g., anendogenous, intracellular enzyme as defined above. The substrate isspecifically converted to a cellular toxin by the intracellular targetenzyme. In another aspect of this invention, the product of an initialprepatory reaction is subsequently activated by a common cellular enzymesuch as an acylase, phosphatase or other “housekeeping” enzyme. Voet, etal. (1995) to release the toxic byproduct from the prodrug.

Further provided by this invention is a method for treating a pathologycharacterized by pathological, hyperproliferative cells in a subject byadministering to the subject a prodrug that is a selective substrate fora target enzyme, and selectively converted by the enzyme to a cellulartoxin in the hyperproliferative cell. The prodrugs of this invention maybe used alone or in combination with other chemotherapeutics oralternative anticancer therapies such as radiation.

A further aspect of this invention is the preparation of a medicamentfor use in treating a pathology characterized by pathological,hyperproliferative cells in a subject by administering to the subject aprodrug that is a selective substrate for a target enzyme, andselectively converted by the enzyme to a cellular toxin in thehyperproliferative cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the development of resistance to anti-cancer modalities incells, and the consequences.

FIG. 2 schematically shows activation pathways of the prodrugs of thisinvention.

FIG. 3 schematically shows the High Throughput Screen for prodrugsactivated by intracellular enzymes important in drug resistance.

FIG. 4 schematically shows how lead human thymidylate synthase (TS)prodrugs are assayed using TS-negative E. coli as the cell target.

FIG. 5 shows an example of how to use this screen to simultaneouslyoptimize the prodrug for reactivity to two target enzymes.

DETAILED DESCRIPTION OF THE INVENTION

The invention is achieved by exploiting some of the key genomic andphenotypic changes intimately linked to resistance to biologic andchemotherapy of cancer cells. The invention provides a means for in vivoselectively inhibiting the growth and/or killing of cells which haveundergone selection by exposure to cancer therapy (including biologictherapy such as tumor necrosis factor (TNF) or chemotherapy). (Refer toTable 1). As a result, certain enzymes which have been activated bymutation or gene amplification are resistant to further therapy by theagent. Unlike prior art therapies directed to creating more potentinhibitors of endogenous, intracellular enzymes, this invention exploitsthe higher enzyme activity associated with therapy-resistant diseasedcells and tissues versus normal cells and tissues and does not rely oninhibiting the enzyme. In one aspect, the tumor cells successfullytreated by the prodrugs of this invention are characterized by enhancedtarget enzyme activity and therefore have a much higher potential toconvert the prodrug to its toxic form than do normal cells which do notoverexpress the target enzyme. The term “target enzyme” is used hereinto define enzymes having one or more of the above noted characteristics.

As used herein, the terms “host cells, “target cells” and“hyperproliferative cells” encompass cells characterized by theactivation by genetic mutation or the endogenous overexpression of anintracellular enzyme. In some embodiments, the overexpression of theenzymes is related to drug resistance or the genetic instabilityassociated with a pathological phenotype. A number of cellularmechanisms are involved in drug resistance, e.g., altered metabolism ofthe drug, impermeability of the cell with regard to the active compoundor accelerated drug elimination from the cell, altered specificity of aninhibited enzyme, increased production of a target molecule, increasedrepair of cytotoxic lesions, or the bypassing of an inhibited reactionby alterative biochemical pathways. Enzymes activated or overexpressedan related to drug resistance include, but are not limited tothymidylate synthase (TS) (Lönn, U. et al. (1996); Kobayashi, H. et al.(1995); Jackman, A. L. et al. (1995)), dihydrofolate reductase(Banerjee, D. et al. (1995) and Bertino, J. R. et al. (1996)), tyrosinekinases (TNF-α, Hudziak, R. M. et al. (1988)) and multidrug resistance(Stühlinger, M. et al. (1994)); Akdas, A. et al. (1996); and (Tannock,I. F. (1996)); and ATP-dependent multidrug resistance associatedproteins (Simon, S. M. and Schindler, M. (1994)). Alternatively,resistance to one drug may confer resistance to other, biochemicallydistinct drugs. While this application is specifically directed tocancer, a similar approach can be applied to enzymes encoded by humanand animal pathogens, and in which the inhibitors have failed due todevelopment of resistance.

Amplification of certain genes is involved in resistance tochemotherapy. Amplification of dihydrofolate reductase (DHFR) is relatedto resistance to methotrexate while amplification of the gene encodingthymidylate synthase is related to resistance to tumor treatment with5-fluoropyrimidnes. Amplification of genes associated with drugresistance can be detected and monitored by a modified polymerase chainreaction (PCR) as described in Kashini-Sabet, et al. (1988) or U.S. Pat.No. 5,085,983. Acquired drug resistance can be monitored by thedetection of cytogenetic abnormalities, such as homogeneous chromosomestaining regions and double minute chromosomes both of which areassociated with gene amplification. Alternative assays include direct orindirect enzyme activity assays and both of which are associated withgene amplification (e.g., Carreras & Santi (1995)); other methodologies(e.g. polymerase chain reaction, Houze, T. A. et al. (1997) orimmunohistochemistry (Johnson, P. G. et al. (1997)).

Alternatively, the target cell is characterized as having inactivatedtumor suppressor function, e.g. loss or inactivation of retinoblastoma(RB) or p53, known to enhance expression of TS (Li, W. et al. (1995)) orDHFR (Berino, et al. (1996) and Li, W. et al. (1995)).

The prodrugs of this invention are useful to treat or ameliorate anydisease wherein the disease-associated enzyme is associated with drugresistance to a chemotherapeutic and in some embodiments, where theenzyme is overexpressed over-accumulated or activated in pathologicalcells versus normal cells, for example, the TS enzyme. Particularlyexcluded is the enzyme glutathione-S-transferase which has been shown tobe elevated in some human tumors. Morgan, A. S. et al. (1998). Theprodrugs of the subject invention are distinguishable on the basis thatthe target enzymes of this invention are commonly overexpressed,overaccumulated or activated in pathological cells versus normal cells.The most important principle which distinguishes the current inventionfrom other approaches are:

(1) This invention describes the synthesis of substrates for enzymeslike thymidylate synthase. The overexpressed enzyme will generate toxin,preferentially in diseased cells. Previous approaches have relied oninhibitor. The inhibitors lead to amplified expression of the enzyme,and subsequent resistance to treatment (see, e.g., Lonn, U. et al.(1996).

(2) The current approach is also distinguishable from other“substrate-prodrug” approaches, e.g., the glutathione-S-transferaseenzymes (see, e.g., Morgan, A. S. et al. (1998). The enzymes of the GSTfamily are expressed at increased levels in response to toxic insult tothe cell. The GST family of enzymes have overlapping substratespecificities, which makes it impossible to design a substrate reactivewith only a single species of enzyme with elevated expression in acancer cell (Morgan, A. S. et al. (1998)). Because each of the enzymesof the current invention (e.g., thymidylate synthase, dihydrofolatereductase and thymidyline kinase) is unique with respect to itsstructure and substrate specificity, it is facile to design uniquesubstrates. Several examples of substrates for thymidylate synthase areprovided in the specifications of this application.

(3) In some cases the gene encoding the target enzyme (e.g., thymidylatesynthase) may have undergone mutation to give resistance to inhibitors,but will still be capable of carrying out reaction with non-inhibitorsubstrates. Barbour, K. W. et al. (1992) and Dicken, A. P. et al.(1993).

Drug Assay

This invention provides a method for identifying agents which havetherapeutic potential for the treatment of hyproliferative or neoplasticdisorders, e.g., cancer. The method also identifies agents that inhibitthe growth of cells or cell cycling of hyperproliferative cells, such ascancer cells. Other cells that are included are bacterial, yeast andparasitic cells which cause disease as a result of inappropriateproliferation in the patient. The agent is considered a potentialtherapeutic agent if cell proliferation, replication or cell cycling isreduced relative to the cells in a control sample. Most preferably, thecells are killed by the agent. The cells can be procaryotic (bacterialsuch as E. coli) or eucaryotic. The cells can be mammalian ornon-mammalian cells, e.g., mouse cells, rat cells, human cells, fungi(e.g., yeast) or parasites (e.g., Pheumocysti or Leishmania) which causedisease.

As used herein, a “hyperproliferative cell” is intended to include cellsthat are de-differentiated, immortalized neoplastic, malignant,metastatic or transformed. Examples of such cells include, but are notlimited to a sarcoma cell a leukemia cell, a carcinoma cell, or anadenocarcinoma cell. More specifically, the cell can be a breast cancercell, a hepatoma cell, a colorectal cancer cell, pancreatic carcinomacell, an oesophageal carcinoma cell, a bladder cancer cell, an ovariancancer cell, a skin cancer cell, a liver carcinoma cell, or a gastriccancer cell. In an alternative embodiment, the target cell can beresistant to a drug or compound used to prevent or kill a cell infectedwith an infectious agent which is resistant to conventional antibiotics.Infectious agents include bacteria, yeast and parasites, such astrypanosomes.

Specific examples of target enzymes that are the subject matter of thisinvention are listed in Table 1 (above) or Table 2 (below). Theseenzymes are involved in resistance to chemotherapy, are endogeneouslyactivated, overexpressed or over-accumulated in a cell characterized byresistance to cancer therapy and associated with a pathological ordisease include, but are not limited to enzymes such as a member of thetyrosine kinase superfamily or an ATP dependent MDR-associated protein,CAD, thymidylate synthase, dihydrofolate reductase, and ribonucleotidereductase. Table 2 provides a list of enzymes which may be targeted bythis approach in infectious disese. TABLE 2 Enzymes Overexpressed ininfectious disease, and which contribute to drug resistance EnzymeProvides increased Resistance to: Beta-lactamases Penicillin and otherbeta-lactam containing antibiotics Aminoglycosidase, or Aminoglycosideantibiotics aminoglycoside midifying enzymes (e.g., streptomycin,gentamycin) Chloramphenicol transacetylase Chloramphenicol TrimethoprimDihydrofolate reductaseReference:Mechanisms of Microbial Disease, 2^(nd) Ed., M. Schaechter, G. Medloff,B. I. Eisenstein, Editor TS Satterfield. Publ. Williams and Wilkins, pp.973 (1993).

The potentially therapeutic agent identified by the method of thisinvention is a prodrug that is a substrate for the enzyme and isconverted intracellularly to an intacellular toxin. As used herein, a“prodrug” is a precursor or derivative form of a pharmaceutically activeagent or substance that is less cytotoxic to target orhyperproliferative cells as compared to the drug metabolite and iscapable of being enzymatically activated or converted into the moreactive form (see Connors, T. A. (1986) and Connors, T. A. (1996)). Thetoxicity of the agent is directed to cells that are producing theconverting enzyme in an amount effective to produce a therapeuticconcentration of the cellular toxin in the diseased cell.

This invention also provides a quick and simple assay that will enableinitial identification of compounds with at least some of the desiredcharacteristics. For purposes of this current invention, the generalscheme of one embodiment is shown in FIG. 3. This drawing describes howthe assay is arranged and the materials necessary for its process. Asshow in FIG. 3, the assay requires two cell types, the first being acontrol cell in which the target enzyme is not expressed, or isexpressed at a low level. The second cell type is the test cell, inwhich the target enzyme is expressed at a detectable level e.g., a highlevel. For example, a procaryotic E. coli which does not endogenouslyexpress the target enzyme TS is a suitable host cell or target cell. Thecell can have a control counterpart (lacking the target enzyme), or in aseparate embodiment, a counterpart genetically modified todifferentially express the target enzyme, or enzymes (containing theappropriate species of target enzyme). More than one species of enzymecan be used to separately transduce separate host cells, so that theeffect of the candidate drug on a target enzyme can be simultaneouslycompared to its effect on another enzyme or a corresponding enzyme fromanother species.

In another embodiment, transformed cell lines, such as ras-transformedNIH 3T3 cells (ATCC, 10801 University Blvd., Manassas, Va. 20110-2209,U.S.A.) are engineered to express variable and increasing quantities ofthe target enzyme of interest from cloned cDNA coding for the enzyme.Transfection is either transient or permanent using procedures wellknown in the art and described in Chen, L. et al. (1996), Hudziak, R. M.et al. (1988), or Carter, P. et al. (1992). Suitable vectors forinsertion of the cDNA are commercially available from Stratagene, LaJolla, Calif. and other vendors. The level of expression of enzyme ineach transfected cell line can be monitored by immunoblot and enzymeassay in cell lysates, using monoclonal or polyclonal antibodypreviously raised against the enzyme for immunodetection. See, e.g., asdescribed by Chen, L. et al. (1996). The amount of expression can beregulated by the number of copies of the expression cassette introducedinto the cell or by varying promoter usage. Enzymatic assays also can beperformed as reviewed by Carreras, C. W. and Santi, D. V. (1995).

As noted above, cells containing the desired genetic deficiencies may beobtained from Cold Spring Harbor, the Agricultural Research ServiceCulture Collection, or the American Type Culture Collection. Theappropriate stains can also be prepared by inserting into the cell agene coding for the target enzyme using standard techniques as describedin Miller (1992), Sanbrook, et al. (1989), and Spector, et al. (1997).Growth assays can be performed by standard methods as described byMiller (1992) and Spector, et al. (1997).

It should be understood by those skilled in the art that the screenshown in FIG. 3 can be applied broadly for the discovery of antibiotics.For example, thymidylate synthase from yeast could be substituted forthat of E. coli in FIG. 4. This would allow the discovery of specificantifungal antibiotics targeting yeast related pathogens. In addition,other enzymes can be subjected to this. For example, prodrugs whichtarget specifically the dihydrofolate reductase activity of infectiousagents, like Pneumocystis carnii, could be selected. These agents willbe selected for specificity for the target enzyme, and can be shown notto activate the enzyme of the natural host by employing the screeningassay described in FIG. 3. The control cellular constructs would containthe corresponding normal human enzyme, in order to show lack of toxicitywhen only the normal human enzyme is present.

For example and as shown in FIG. 4, a foreign gene, e.g., a human geneencoding TS, can be inserted into the host cell such that human TS isexpressed. This genetically engineered cell is shown as the “test cell”in FIG. 3. The “control cell” does not express the target enzyme. Insome embodiments it may be necessary to supplement the culture mediawith the protein product of the target enzyme.

In a separate embodiment, the wild type host cell is deficient or doesnot express more than one enzyme of interest. As shown in FIG. 4, thehost cell does not endogenously produce thymidine kinase (TK⁻) orthymidylate synthase (TS⁻). Genes coding for the human counterpart ofthese enzymes are introduced into the host cell to obtain the desiredlevel of expression. The level of expression of enzyme in eachtransfected cell line can be monitored by methods described herein,e.g., by immunoblot and enzyme assay in cell lysates, using monoclonalor polyclonal antibody previously raised against the enzyme forimmunodetection. See, e.g., as described by Chen, L. et al. (1996).Enzymatic assays also can be performed as reviewed by Carerras, C. W.and Santi, D. N. (1995) using detectably labeled subsituents, e.g.tritium labeled substitutes.

The test cell is grown in small multi-well plates and is used to detectthe biologic activity of test prodrugs. For the purposes of thisinvention, the successful candidate drug will block the growth or killthe test cell type, but leave the control cell type unharmed.

The candidate prodrug can be directly added to the cell culture media orpreviously conjugated to a ligand specific to a cell surface receptorand then added to the media. Methods of conjugation for cell specificdelivery are well known in the art, see e.g., U.S. Pat. Nos. 5,459,127;5,264,618; and published patent specification WO 91/17424 (publishedNov. 14, 1991). The leaving group of the candidate prodrug can bedetectably labeled, e.g., with tritium. The target cell or the culturemedia is then assayed for the amount of label released from thecandidate prodrug. Alternatively, cellular uptake may be enhanced bypackaging the prodrug into liposomes using the method described inLasic, D. D. (1996) or combined with cytofectin as described in Lewis,J. G. et al. (1996).

In a separate embodiment, cultured human tumor cells overexpressing theenzyme of interest i.e., target enzyme, are identified as describedabove. The cells are contacted with the potential therapeutic agentunder conditions which favor the incorporation of the agent into theintracellular compartment of the cell. The cells are then assayed forinhibition of cellular proliferation or cell killing.

Provided below is a brief summary of cells and target enzymes that areuseful to activate the prodrugs of this invention.

Thymidylate Synthase

The overexpression of thymidylate synthase is associated with coloncancer, breast cancer, gastric cancer, head and neck cancer, livercancer and pancreatic cancer. These diseases are currently treated byantimetabolite drugs (uracil-based, folate-based, or quinaszoline-based(see Table 1)). In each of these cases it is likely that the5-fluorouracil therapy can lead to amplified activity of TS, or selectfor drug resistant forms of the enzyme, and thereby lead todrug-resistance of the disease relapse. Lönn, U. et al. (1996) reportedthat amplification of the TS gene occurred in breast cancer patients whopreviously received adjuvant chemotherapy (cyclophosphamide,methotrexate, 5-fluomuracil [CMF]) after surgery. The principal reactionnormally performed by TS is the synthesis of deoxythymidinemonophosphate (dTMP) and dihydrofolate (DH) from deoxyuridinemonophosphate (dUMP) and N(5),N(10)methylene-tetrahydrofolate (THF). Inone embodiment, a derivative of uracil or THF is provided to cellsexpressing TS. For purposes of this invention, “uracil” (base only) and“uridine” (base and sugar) are used interchangeably and synonymously.

The derivative or “prodrug” is converted by the enzyme into highlycytotoxic metabolites. The low level of TS expressed in normal cellswill not produce a toxic amount of the converted toxin. High levels ofTS expressed in disease tissues generate more toxin and thereby lead toan inhibition of cell proliferation and/or cell death. For example,current therapy utilizes 5-fluorodeoxyuridylate to inhibit TS activity.During the reaction with substrate, the fluorine atom irreversibly bindsto the TS enzyme and inhibits it. In contrast to one embodiment of thepresent invention, the prodrugs allow TS to complete the reaction butgenerates a modified product that, when incorporated into DNA, canes atoxic effect. The enzyme product may also block other critical cellularfunctions (e.g. protein synthesis or energy metabolism). Conversion ofthe prodrug also can release a metabolite, such as Br⁻ or I⁻ or CN⁻which is toxic to the cell. Derivatives of uracil/dUMP and N(5)(10)-THFcan be synthesized all of which have the potential of generating toxicproduct after metabolically catalyzed by TS.

Synthesis of 5-substituted pyrimidine nucleosides and 5-substitutedpyrimidine nucleoside monophosphats can be accomplished by methods thatare well-known in the art. For example, treatment of5-chloromercuri-2′-deoxyuridine with haloalkyl compounds, hoaces orhaloalkenes in the presence of Li₂PdCl₄ results in the formation,through an organopalladium intermediate, of the 5-alkyl, 5-acetyl or5-alkene derivative, respectively. Wataya, et al. (1979) and Bergstrom,et al. (1981). Another example of C5-modification of pyrimidinenucleosides and nucleotides is the formation of C5-trans-styrylderivatives by treatment of unprotected nucleotide with mercuric acetatefollowed by addition of styrene or ring-substituted styrenes in thepresence of Li₂PdCl₄. Bigge, et al. (1980). Pyrimidinedeoxyribonucleoside triphosphates were derivatize with mercury at the 5position of the pyridine ring by treatment with mercuric acetate inacetate buffer at 50° for 3 hours. Dale, et al. (1973). Such treatmentwould also be expected to be effective for modification ofmonophosphates; alteratively, a modified triphosphate could be convertedenzymatically to a modified monophosphate, for example, by controlledtreatment with alkaline phosphatase followed by purification ofmonophosphate. Other moieties, organic or nonorganic, with molecularproperties similar to mercury but with preferred pharmacologicalproperties could be substituted. For general methods for synthesis ofsubstituted pyrimidines, for example, U.S. Pat. Nos. 4,247,544;4,267,171; and 4,948,882; and Bergstom et al. (1981). The above methodswould also be applicable to the synthesis of derivatives of5-substituted pyrimidine nucleosides and nucleotides containing sugarsother than ribose or 2′-deoxyribose, for example 2′-3′-dideoxyribose,arabinose, furanose, lyxose, pentose, hexose, heptose, and pyranose. Anexample of such a subsituents are halovinyl groups, e.g.E5-(2-bromovinyl)-2′-deoxyuridylate. Barr, F. J. et al. (1983). In thisreference the authors demons that the normally inert substituent at the5-position (bromovinyl) is convertible to a chemically reactive group asa result of enzyme-mediated nucleophilic attack at the 6-position of theuridine heterocycle, leading to the production of a reactive alkylatingagent. This compound is not useful from the point of view of the currentapplication because it cannot be activated by endogenous thymidinekinase, and because its conversion by thymidylate synthase leads toinactivation of the thymidylate synthase (Balzarini, et al., 1987).However, improved subsituents will be synthesized and compared forreactivity with TS and specific cytotoxicity to TS-overproducing tumorcells.

Alternatively, 5-bromodeoxyuridine, 5-iododeoxyuridine, and theirmonophosphate derivatives are available commercially firm Glen Research,Stering, Va. (USA), Sigma-Aldrich Corporation, St. Louis, Mo. (USA),Moravek Biochemicals, Inc., Brea, Calif. (USA), ICN, Costa Mesa, Calif.(USA) and New England Nuclear, Boston, Mass. (USA).Commercially-available 5-bromodeoxyuridine and 5-iododeoxyuridine can beconverted to their monophosphates either chemically or enzymatically,though the action of a kinase enzyme using commercial available reagentsfrom Glen Research, Sterling, Va. (USA) and ICN, Costa Mesa, Calif.(USA). These halogen derivatives could be combined with othersubsituents to create novel and more potent antimetabolites.

Primary sequences show that TS is one of the most highly conservedenzymes. Perry, K. et al. (1990). Crystal structures of TS from severalprocaryotic species, Lactobacillus casei (Hardy, L. W. et al. (1987);Finer-Moore, J. et al. (1993)) and Escherichia coli (Perry, K et al.(1990)); an eukaryote Leishmania major (Knighton, E. R. et al. (1994));and T4 phage (Finer-Mooze, J. S. et al., (1994)) have been determinedand indicate that tertiary structure also is very well conserved. Thesequence alignment of the species of TS whose three dimensionalstructures have been determined and is shown in Schiffer, C. A. et al.(1995). From these amino acid sequences, the DNA sequences can bededuced or isolated using methods well known to those of skill in theart. Sambrook, et al. (1989). Alternatively, some 29 TS sequences fromdifferent organisms have been cloned and deposited into the DNAdatabases as described in Carerras, C. W. and Santi, D. V. (1995). Thesequence of human thymidylate synthase gene, its cloning, expression andpurification is provided in Takeishi, K. et al. (1985), Davisson, V. J.et al. (1989) and Davisson, V. J. et al. (1994). Genes encoding the TSprotein and containing the necessary regulatory sequences, areconstructed using methods well known to those of skill in the art. Thegene encoding TS is introduced to target cells by electroporation,transformation or transfection procedures. Sambrook et al. (1989).Alternatively, the gene is inserted into an appropriate expressionvector by methods well known in the art, e.g., as described in Carreras,C. W. and Santi, D. V. (1995), Miller (1992) and Spector et al. (1997).The expression vector inserts the TS gene into the cells. The cells arethen grown under conditions which favor the expression and production ofTS protein.

Human gastric cancer cell lines, MKN-74, MKN-45, MKN-28 and KATO-III canbe used in the assay described above to identify potential therapeuticagents which are selective substrates for TS. MKN-74 and MKN-45 areestablished from well and poorly differentiated adenocarcinomas,respectively. These cell lines and culture conditions are described inOsmki M. et al. (1997) and references cited therein. Alternatively,tumor cell lines such as those described by Copur, S. et al. (1995),which have been selected by 5-FU to overexpress thymidylate synthase maybe used.

Quantitation of TS can be performed using epic biochemical assays thatare well known to those with skill in the art. To quantify the level ofTS protein and TS gene expression from human tumor tissue samples, themethods as reported by Johnston, P. G. et al. (1991) and Horikoshi, T.et al. (1992) provide sensitive assays. Alternatively, the PCR method ofLönn, U. et al. (1996) is used to assay TS gene amplification andidentify cells that are useful in the method of identifying therapeuticagents as described herein.

As is apparent to one skilled in the art control cell culture systemswithout drug and separately with a reference drug such as the oneexemplified below, also am assayed. A preferred embodiment of theprodrugs is one which preferentially kills target cells with about2-fold and preferably about 3-fold or greater activity than normalcells. This invention also provides the agents identified by the methodsdescribed herein.

In another aspect, this invention provides a method for inhibiting theproliferation of a hyperproliferative cell, by first conducting theabove assay. A prodrug identified by this assay is contacted with thecell and converted to a toxic metabolite in the cell by an endogenousintacellular enzyme as described above.

In one embodiment, the endogenous, intracellular enzyme is thymidylatesynthase and the cell is selected from the group consisting ofcolorectal cell, head and neck cancer cell, breast cancer cell, or agastric cancer cell.

In a further aspect, the prodrug contacted with the cell overexpressingthymidylate synthase is an L- or D compound of the formulae:

which may be in any of their enantiomeric, diasteriomeric, orstereoisomeric forms, including, for example, D- or L-forms, and forexample, α- or β-anomeric forms.

In the above formulae, R₁ (at the 5-position) is or contains a leavinggroup which is a chemical entity that has a molecular dimension andelectrophilicity compatible with extraction from the pyrimidine ring bythymidylate synthase, and which upon release from the pyrimidine ring bythymidylate synthase, has the ability to inhibit the proliferation ofthe cell or kill the cell.

In one embodiment, R₁ is or contains a chemical entity selected from thegroup consisting of: —Br, —I, -O-alkyl, -O-aryl, O-heteroaryl, -S-alkyl,-S-aryl, -S-heteroaryl, —CN, —OCN, —SCN, —NH₂, -NH-alkyl, -N(alkyl)₂,—NHCHO, —NHOH, -NHO-alkyl, NH₂CONHO—, NHNH₂, and —N₃. Another example ofR₁ is derived from cis-platin:

In the above formulae for the L- or D-compound(s), Q is a chemicalentity selected from the group consisting of sugar groups, thio-sugargroups, carbocyclic groups, and derivatives thereof. Examples of sugargroups include, but are not limited to, monosaccharide cyclic sugargroups such as those derived from oxetanes (4-membered ring sugars),furanoses (5-membered ring sugars), and pyranoses (6-membered ringsugars). Examples of furanosyl include threo-furanosyl (from threose, afour-carbon sugar); erythro-furanosyl (from erythrose, a four-carbonsugar); ribo-furanosyl (from ribose, a five-carbon sugar); ara-furanosyl(also often referred to as arabino-furanosyl; from arabinose, afive-carbon sugar); xylo-furanosyl (from xylose, a five-carbon sugar);and lyxo-furanosyl (from lyxose, a five-carbon sugar). Examples of sugargroup derivatives include “deoxy”, “keto”, and “dethydro” derivatives aswell as substituted derivatives. Examples of thio sugar groups includethe sulfur analogs of the above sugar groups, in which the ring oxygenhas been replaced with a sulfur atom. Examples of carbocyclic groupsinclude C₄ carbocyclic groups, C₅ carbocyclic groups, and C₆ carbocyclicgroups which may further have one or more subsituents, such as —OHgroups.

In one embodiment, Q is a furanosyl group of the formula:

wherein R₂ and R₃ arc the same or different and are independently H or—OH. In one embodiment, R₂ and R₃ are H. In one embodiment, R₂ is OH andR₃ is H. In one embodiment, R₂ is H and R₃ is OH. In one embodiment,where R₂ and R₃ are OH.

In one embodiment, Q is a β-D-ribofuranosyl group of the formula:

wherein R₂ and R₃ are the same or different and are independently H or—OH.

In some embodiments the hydroxymethyl group (for example, the4′-hydroxymethyl group of β-D-ribofuranosyl) can be phosphorylated.

Modifications of current alkylating agents attached at the pyrimidine5-position, and which fit the steric restrictions as described above canbe employed (Haskell, C. M. eds. (1995), pp. 55-58). Cell-free, or cellbased, screening assays for release of constituent at the 5-position ofuracil are described by Roberts, D. (1966) and Hasimoto, Y. et al.(1987).

In the case where R₁ comprises CN⁻, the highly toxic CN⁻ moiety is thetherapeutically active species. Because of the highly nonspecific toxicnature of CN⁻, it cannot normally be used in a therapeutic mode. Thisproblem is overcome by delivering the toxin in the form of a prodrugthat will be significantly activated only in cells which overexpressthymidylate synthase.

In addition, a prodrug can be converted to a toxic metabolite by theintracellular enzyme which, in some embodiments, can be further modifiedby an intracellular “housekeeping” enzyme. An example is shown below.

Description of the “partial” reaction of dUMP and TS, as well asrelevant assays are described in Garett, C. et al. (1979). Assays forother product, i.e. where a reaction complete product is ananti-metabolite of the bromovinyl-derivatives of dUNP, are described byBarr, P. J., et al. (1983). Salts of the prodrug of the presentinvention may be derived from inorganic or organic acids and bases.Examples of acids include hydrochloric, hydrobromic, sulfuric, nitric,perchloric, fumaric, maleic, phosphoric, glycollic, lactic, salicyclic,succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic andbenzenesulfonic acids. Other acids, such as oxalic, while not inthemselves pharmaceutically acceptable, can be employed in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable acid additionsalts. Examples of bases include alkali metal (e.g., sodium) hydroxides,alkaline earth metal (e.g., magnesium) hydroxides, ammonia, andcompounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl.

Examples of salts include; acetate, adipate, alginate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camposulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, malcate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylproprionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate and undecanoate. Otherexamples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group).

For therapeutic use, salts of the compounds of the present inventionwill be pharmaceutically acceptable. However, salts of acids and baseswhich are non-pharmaceutically acceptable may also find use, forexample, in the preparation or purification of a pharmaceuticallyacceptable compound.

Esters of the prodrugs or compounds identified by the method of thisinvention include carboxylic acid test (i.e., —O—C(═O)R) obtained byesterification of the 2′-, 3′- and/or 5′-hydroxy groups, in which R isselected from (1) straight or branched chain alkyl (for example,n-propyl, t-butyl, or n-butyl), alkoxyalkyl (for example,methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (forexample, phenoxymethyl), aryl (for example, phenyl optionallysubstituted by, for example, halogen, C₁₋₄alkyl, or C₁₋₄alkoxy oramino); (2) sulfonate esters such as alkylsulfonyl (for example,methanesulfonyl) or aralkylsulfonyl; (3) amino acid esters (for example,L-valyl or L-isoleucyl); (4) phosphonate esters and (5) mono-, di- ortriphosphate esters. The phosphate esters may be further esterified by,for example, a C₁₋₂₀ alcohol or reactive derivative thereof, or by a2,3di-(C₆₋₂₄)acyl glycerol. In such esters, unless otherwise specified,any alkyl moiety present advantageously-contains from 1 to 18 carbonatoms, particularly from 1 to 6 carbon atoms, more particularly from 1to 4 carbon atoms. Any cycloalkyl moiety present in such estersadvantageously contains from 3 to 6 carbon atoms. Any aryl moietypresent in such esters advantageously comprises a phenyl group. Examplesof lyxo-furanosyl prodrug derivatives of the present invention include,for example, those with chemically protected hydroxyl groups (e.g., withO-acetyl groups), such as 2′-O-acetyl-lyxo-furanosyl;3′-O-acetyl-lyxo-furanosyl; 5′-O-acety-lyxo-furanosyl;2′,3′-di-O-acetyl-lyxo-furanosyl and2′,3′,5′-ti-O-acetyl-lyxo-furanosyl.

Ethers of the compounds of the present invention include methyl, ethylpropyl, butyl, isobutyl and sec-butyl ethers.

In a further embodiment, the substrate may not be chemically related topyrimidines or folates, but rather synthesized based upon knownparameters of rational drug design. See Dunn, W. J. et al. (1996).

As is apparent to one skilled in the art, control cell culture systemswithout drug and separately with a reference drug such as the oneexemplified below, also are assayed. Compounds which preferentially killtarget cells with about 2-fold and preferably 3-fold or greater activitythan normal cells are preferred. This invention also provides the agentsidentified by the methods described herein.

Tyrosine Kinases

The tyrosine kinase superfamily comprises the EGF receptor (EGFR), themacrophage colony-stimulating factor (CSF-1) receptor (v-fms), and theinsulin receptor, which shows 30 to 40% identity with the product of theros oncogene. More specifically, the members of this superfamily includev-src, c-src, EGFR, HER2, CSF-1 receptor, c-fms, v-ros, insilinreceptor, and c-mos. See FIG. 8.5 of Burck, K. B. et al., eds. (1988).Overexpression of members of the type 1 receptor tyrosine kinasesuperfamily has been documented in many types of cancer (Eccles, S. A.et al. (1994-95)). Overexpression of tyrosine kinases is linked toexposure to the α-cancer biologic agent TNF-α (Hudziak, R. M. et al.(1988) and Hudziak R. M. et al. (1990)) and to chemotherapy (Stühlingeret al. (1994)).

The transforming gene of the Rous sarcoma virus, v-src, encodes anenzyme that phosphoryl tyrsine residues on proteins. The c-srcproto-oncogene is found on chromosome 20. Tissues and cell lines derivedfrom tumors of neuroectodermal origin having a neural phenotype expresshigh levels of c-src accompanied by high specific kinase activity.

Several groups of investigators have reported overexpression ofc-erbB-2/neu (“HER2”) oncogene in cancer cells. Brison (1993) noted thaterbB proto-oncogene is amplified in human tumors with resultantoverexpression in most cases. Amplification of the c-erbB-2/neu oncogenehas been reported in human mammary tumors (Slamon, et al. (1987), van deVijver et al. (1987), Pupa et al. (1993), and Anderson et al. (1995))and in blader tumors (Sauter et al. (1993)), and in every caseamplification was accompanied by overexpression. c-erB-2/neaoverexpression also has been reported in ovarian cancer tissue samples(Slamin, et al. (1989), Meden et al. (1994), and Felip et al. (1995)),and tumors derived from the peripheral nervous system. Sukumar andBarbacid, (1990).

To perform the drug screening assay, tumor cell lines will be assayedfor expression of the oncogene or will be engineered to express varyinglevels of tyrosine kinase. Selected cell lines arm cultured andcandidate drugs are added in varying concentrations. The cells areassayed for cell killing or inhibition of cellular proliferation, asdescribed in Hudziak, R. M. et al. (1988) and Hudziak, R. M. et al.(1990).

Dihydrofolate Reductase

Methotrexate is a potent inhibitor of dihydrofolate reductase, an enzymenecessary for intracellular folate metabolism. Dihydrofolate reductasefunctions to regenerate tetrahydrofolate from dihydrofolate, a productof the thymidylate synthase reaction (Voet, et al. eds. (1995), p. 813).It is was established that an important mechanism of resistance of cellsto methotrexate is an increase in DHFR activity due to amplification ofthe DHFR gene. Banerjee, D. et al. (1995), Schimke, R. T. et al. (1988).Lönn, U. et al. (1996) reported that amplification of the DHFR geneoccurred in breast cancer patients who previously received adjuvantchemotherapy (cyclophosphamide, methotrexate, 5-fluorouracil [CMF])after surgery. Lack of the retinoblastoma (Rb) may also lead to enhancedMTX resistance as a consequence of an increase in DHFR mRNA expressionactivity without gene amplification. Li, W. W. et al. (1995). Cell lineswith mutated p53 have been shown to undergo gene amplification, and theresistant cells are selected by chemotherapy. Banerjee, D. et al.(1995), Yin, Y. et al. (1992) and Livingston, L. R. et al. (1992). Forthe purposes of performing the assay of this invention, Schimke, R. T.et al. (1988) describes several mouse, hamster and human cell lines.Alternatively, the PCR method of Lönn U. et al. (1996) is used to assayDHFR gene amplification and identify cells that are useful in the methodof identifying therapeutic agents as described herein. The nucleotidesequence of the cDNA coding for the human dihydrofolate reductase isprovided in Masters, J. N. and Attardi, G. (1983) and cells can beengineered to express varying levels of the enzyme as noted herein.Dicken, A. P. et al. (1993) describes a mutant DHFR gene selected bychemotherapy. Purification of DHFR and assays related to enzyme functionare described in Nakano, T. et al. (1994). Alternatively, cDNA encodingDHFR is transfected into NIH 3T3 cells. Candidate drugs are added invarying concentrations and cell killing and inhibition of proliferationare assayed.

Antimetabolites dependent on dihydrofolate reductase activity can besynthesized by the attachment of, for example, an alkylating group toeither the N5 or the C6 position of dihydrofolate. Reduction of theN5-C6 bond by DHFR will result in the release of the alkylating agent.In addition to the alkylating groups, any moiety whose release by DHFRresults in the production of a toxin or an antimetabolite will be usefulin the practice of the invention.

Multidrug Resistant Tumors

Multidrug resistance (MDR) is a generic term for the variety ofstrategies tumor cells use to evade the cytotoxic effects of anticancerdrug. MDR is characterized by a decreased sensitivity of tumor cells notonly to the drug employed for chemotherapy but also to a broad spectrumof drugs with neither obvious structural homology nor common targets.This phenotopic resistance is one of the major obstacles to thesuccessful treatment of tumors. MDR may result from structural orfunctional changes at the plasma membrane or within the cytoplasmcellular compartments, or nucleus. Molecular mechanisms of MDR arediscussed in terms of modifications in detoxification and DNA repairpathways, changes in cellular sites of drug sequestration, decreases indrug-target affinity, synthesis of specific drug inhibitors withincells, altered or inappropriate targeting of proteins, and acceleratedremoval or secretion of drugs.

One of the mechanisms implicated in MDR results from amplification andover-expression of a gene known as the ATP-dependent multidrug resistantassociated protein (MRP) in drug selected cell lines. For a review ofthe mechanisms of MDR, see Gottesman, M. M. et al. (1995) and Noder etal. (1996).

To establish MDR cell lines, drug selections are conducted in either asingle step or in multiple steps as described in Gottesman, M. M. et al.(1995) and Simon, S. M. and Schindler, M. (1994), and references citedtherein. The isolation of DNA sequences coding for MDR from variousmammalian species is described in Gros, P. et al. (1986), Gudkov, A. V.et al. (1987), and Roninson, I. B. et al. (1984), and reviewed inGottesman, M. M. et al. (1995), and cells can be engineer to expressvarying levels of this enzyme as described above. The prodrug targetingMDR will be based upon the ATPase activity of this transporter.

Ribonucleotide Reductase

The ribonucleotide reductase reduces ribonucleotide diphosphates to thecorresponding deoxyribonucleoside diphosphates. The enzyme is a tetramermade up of two α-subunits and two β-subunits. Hydroxyurea specificallyblocks this reaction by interacting with the tyrosyl free radical(Tyr-122) of the β₂-substrate complex. Voet et al. (1995). The goal intargeting this reaction is to allow the accumulation of the free radicalproduct O₂ ⁻, which is highly cytotoxic.

Application of Technology to Other Diseases

While the primary focus of this application is directed to cancer, itshould be recognize that the technology is broadly applicable to otherdisease, especially antibiotic resistant bacterial infections. Theβ-lactam antibiotics encounter resistance in bacteria as the result ofoverexpression of β-lactamases. Hamilton-Miller, J. M. T. and Smith, J.T. eds. (1979) p. 443. Other enzymes, such as the aminoglycosidephosphotransferase Type III, a induced and selected for followingtreatment with aminoglycoside antibiotics, such as kanamycin. McKay, G.A. et al. (1994). For the purpose of this application, prodrugsubstrates derived from known substrates will be prepared that will notblock enzyme activity, but will instead take advantage of the highenzyme activity to generate intracellular toxins in the infectiousagents.

In Vivo Administration

The in vitro assays are confirmed in animal models bearing human tumorsor infected with an antibiotic resistant microorganism to determine invivo efficacy.

Another aspect of this invention is a method for treating a pathologycharacterized by hyperliferative cells in a subject comprisingadministering to the subject a therapeutic amount of a prodrug that isconverted to a toxin in a hyperproliferative cell by an endogenousintracellular enzyme as defined herein.

When the prodrug is administered to a subject such as a mouse, a rat ora human patient, the agent can be added to a pharmaceutically acceptablecarrier and systemically or topically administered to the subject. Todetermine patients that can be beneficially treated a tumor sample isremoved from the patient and the cells are assayed for the level ofexpression of the enzyme of interest. If the expression is above thatexpressed in normal cells and an amount of the prodrug effective to killor inhibit the cell can be administered without undesirable sideeffects, then the prodrug is a preferred embodiment. Therapeutic amountscan be empirically determined and will vary with the pathology beingtreated, the subject being treated and the toxicity of the convertedprodrug or cellular toxin. When delivered to an animal, the method isuseful to further confirm efficacy of the prodrug. As an example of ananimal model, groups of nude mice (Balb/co NCR nu/nu female, Simonsen,Gilroy, Calif.) are each subcutaneously inoculated with about 10³ toabout 10⁹ hyperproliferative, cancer or target cells as defined herein.When the tumor is established, the prodrug is administered for example,by subcutaneous injection around the tumor. Tumor measurement todetermine reduction of tumor size are made in two dimensions usingvenier calipers twice a week. Other animal models may also be employedas appropriate. Lovejoy, et al. (1997) and Clarke, R. (1996).

Administration in vivo cam be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration arewell known to those of skill in the art and will vary with thecomposition used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physicians. Suitable dosage formulations andmethods of administering the agents can be found below.

The agents and compositions of the present invention can be used in themanufacture of medicaments and for the treatment of humans and otheranimals by administration in accordance with conventional procedures,such as an active ingredient in pharmaceutical compositions.

The pharmaceutical compositions can be administered orally,intranasally, parenterally or by inhalation therapy, and may take theform of tablets, lozenges, granules, capsules, pills, ampoules,suppositories or aerosol form. They may also take the form ofsuspensions, solutions and emulsions of the active ingredient in aqueousor nonaqueous diluents, syrups, granulates or powders. In addition to acompound of the present invention, the pharmaceutical compositions canalso contain other pharmaceutically active compounds or a plurality ofcompounds of the invention.

More particularly, a compound of the formula of the present inventionalso referred to herein as the active ingredient, may be administeredfor therapy by any suitable mute including oral, rectal, nasal, topical(including transdermal, aerosol buccal and sublingual), vaginal,parental (including subcutaneous, intramuscular, intravenous andintradermal) and pulmonary. It will also be appreciated that thepreferred route will vary with the condition and age of the recipient,and the disease being treated.

In general, a suitable dose for each of the above-named compounds, is inthe range of about 1 to about 100 mg per kilogram body weight of therecipient per day, preferably in the range of about 1 to about 50 mg perkilogram body weight per day and most preferably in the range of about 1to about 25 mg per kilogram body weight per day. Unless otherwiseindicated, all weights of active ingredient are calculated as the parentcompound of the formula of the present invention for salts or estersthereof, the weights would be increased proportionately. The desireddose is preferably presented as two, three, four, five, six or moresub-doses administered at appropriate intervals throughout the day.These subdoses may be administered in unit dosage forms, for example,containing about 1 to about 100 mg, preferably about 1 to above about 25mg, and most preferably about 5 to above about 25 mg of activeingredient per unit dosage form. It will be appreciated that appropriatedosages of the compounds and compositions of the invention may depend onthe type and severity and stage of the disease and can vary from patientto patient. Determining the optimal dosage will generally involve thebalancing of the level of therapeutic benefit against any risk ordeleterious side effects of the treatments of the present invention.

Ideally, the prodrug should be administered to achieve peakconcentrations of the active compound at sites of disease. This may beachieved, for example, by the intravenous injection of the prodrug,optionally in saline, or orally administered for example, as a tablet,capsule or syrup containing the active ingredient. Desirable bloodlevels of the prodrug may be maintained by a continuous infusion toprovide a therapeutic amount of the active ingredient within diseasetissue. The use of operative combinations is contemplated to providetherapeutic combinations requiring a lower total dosage of eachcomponent antiviral agent than may be required when each individualtherapeutic compound or drug is used alone, thereby reducing adverseeffects.

While it is possible for the prodrug ingredient to be administeredalone, it is preferable to present it as a pharmaceutical formulationcomprising at least one active ingredient, as defined above, togetherwith one or more pharmaceutically acceptable carriers therefor andoptionally other therapeutic agents. Each carrier must be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not injurious to the patient.

Formulations include those suitable for oral, rectal, nasal, topical(including transdermal, buccal and sublingual), vaginal, perenteral(including subcutaneous, intramuscular, intravenous and intradermal) andpulmonary administration. The formulations may conveniently be presentedin unit dosage form and may be prepared by any methods well known in theart of pharmacy. Such methods include the step of bringing intoassociation the active ingredient with the carrier which constitutes oneor more accessory ingredients. In general, the formulations art preparedby uniformly and intimately bringing into association the activeingredient with liquid carriers or finely divided solid carriers orboth, and then if necessary shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tablets,contain a predetermined amount of the active ingredient; as a powder orgranules; as a solution or suspension in an aqueous or non-aqueousliquid; or as an oil-in-water liquid emulsion or a water-in-oil liquidemulsion. The active ingredient may also be presented a bolus, electuaryor paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in a fee-flowingfrom such as a powder or granules, optionally mixed with a binder (e.g.,povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inertdiluent, preservative, disintegrate (e.g., sodium starch glycolate,crosslinked povidone, cross-linked sodium carboxymethyl cellulose)surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein using, for example,hydroxypropylmethyl cellulose in varying proportions to provide thedesired release profile. Tablets may optionally be provided with anenteric coating, to provide release in pans of the gut other than thestomach

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Pharmaceutical compositions for topical administration according to thepresent invention may be formulated as an ointment, cream, suspension,lotion, powder, solution, paste, gel, spray, aerosol or oil.Alternatively, a formulation may comprise a patch or a dressing such asa bandage or adhesive plaster impregnated with active ingredients andoptionally one or more excipients or diluents.

For diseases of the eye or other external tissue, e.g., mouth and skin,the formulations are preferably applied as a topical ointment or creamcontaining the active ingredient in an amount of for example, about0.075 to about 20% w/w, preferably about 0.2 to about 25% w/w and mostpreferably about 0.5 to about 10% w/w. When formulated in an ointment,the prodrug may be employed with either a paraffinic or a water-miscibleointment base. Alteratively, the prodrug ingredients may be formulatedin a cream with an oil-in-water cream base.

If desired, the aqueous phase of the cream base may include, forexample, at least about 30% w/w of a polyhydric alcohol, i.e., analcohol having two or more hydroxyl groups such as propylene glycol,butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycoland thereof. The topical formulations may desirably include a compoundwhich enhances absorption or penetration of the prodrug ingredientthrough the skin or other affected areas. Examples of such dermalpenetration enhance include dimethylsulfoxide and related analogues.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in an known manner. While this phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at lease one emulsifier with a fat or an oil orwith both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make up theso-called emulsifying wax, and the wax together with the oil and/or fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the present invention include Tween 60, Span 80, cetosearyl alcohol,myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties, the solubility of the activecompound in most oils likely to be used in pharmaceutical emulsionformulations is very low. Thus the cream should preferably be anon-greasy, non-staining and washable product with suitable consistencyto avoid leakage from tubes or other containers. Straight or branchedchain mono- or dibasic alkyl esters such as di-isoadipate, isocetylstearate, propylene glycol diester of coconut fatty acids, isopropylmyristate, decyl oleate, isopropyl palmitate, butyl irate, 2-ethylhexylpalmitate or a blend of blanched chain esters known as Crodamol CAP maybe used, the last three being preferred esters. These may be used aloneor in combination depending on the properties required. Alternatively,high melting point lipids such as white soft paraffin and/or liquidparaffin or other mineral oils can be used.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the prodrugingredient. The prodrug ingredient is preferably present in suchformulation in a concentration of about 0.5 to about 20%, advantageouslyabout 0.5 to about 10% particularly about 1.5% w/w.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for vagina administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the prodrug ingredient, such carriers as areknown in the art to be appropriate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of about 20 to about 500 microns which is administered in themanner in which snuff is taken, i.e., by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid for administrationas, for example, nasal spray, nasal drops, or by aerosol administrationby nebulizer, include aqueous or oily solutions of the prodrugingredient.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. The formulations may be presented in unit-dose ormulti-dose sealed contains, for example, ampoules and vials, and may bestored in a freeze dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, ganules andtablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily subdose, as herein above-recited, or an appropriate factionthereof, of a prodrug ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the an having regard to the type of formulationin question, for example, those suitable of oral administration mayinclude such further agents as sweeteners, thickeners and flavoringagents.

Prodrugs and compositions of the formula of the present invention mayalso be presented for the use in the form of veterinary formulations,which may be prepared, for example, by methods that are conventional inthe art.

EXAMPLES

The following examples are specifically directed to the target enzymeTS. It is apparent to those skilled in the art that the followingmethods can be modified for the discovery of other prodrugs to targetenzymes as defined herein.

Chemical and Cell-Based Assays

Pyrimidine-based prodrugs are chosen based on the ability to react withintracellular thymidylate synthase, and release the toxin into themedium without containing the enzyme. Candidate drug are screened inreaction mixtures containing human thymidylate synthase with and withoutN5N10-methylenetetrahydrofolate, and the candidate prodrug. The leavinggroup of the candidate prodrug (e.g., at the pyrimidine 5 position) islabeled, for example, with tritium using methods well known in the art.The control substrate is similarly labeled (e.g. 5-³H) dUMP, under thesame reaction conditions. The assays are done similarly to thedescription provided in Carreras, C. W. and Santi, D. V. (1995), andreferences cited therein. The human thymidine synthase can be purifiedfrom E. coli containing the expressed human thymidylate synthase. SeeDavisson, V. J. et al. (1989) and Davisson, V. J. et al. (1994). Thisapproach provides a scaleable assay capable of screening large numbersof candidate compounds.

A high throughput screen to identify biologically active compounds isoutlined in FIGS. 3, 4 and 5. The basis of the test is the ease ofgenetic manipulation and growth of E coli, and similar single cellorganisms (e.g. yeast), see Miller (1992) and Spector, et al. (1997).The key step is g the endogenous enzyme activity corresponding to anenzyme target for prodrug design. This can be done by any of the methodsdescribed by Miller (1992), Sambrook (1989) or Spector et al. (1997).These methods include chemical and biologic (e.g. viral or t oninsertional) mutagenesis, followed by an appropriate selection procedureThe TS negative (TS) cell then becomes a negative control for theidentification of prodrugs that, when acted upon by thymidylatesynthase, become cell toxins. A similar approach can be made with othercell types, e.g. other bacteria, yeast or other selectable single cellorganisms. In the assay, both control and recombinant organisms arecompared for sensitivity to the test compounds. As will be understood bythose skilled in the art, prodrugs which distinguish between species ofenzyme can also be derived from this procedure. For example, otherwiseidentical cells expressing hunk and yeast enzymes can be used to detectantibiotic prodrugs which are preferentially toxic only to the cellsexpressing the yeast enzyme. In this way, novel and specific antibioticscan be discovered.

Example cell lines are ras-transformed NIH 3T3 cells (obtained from theATCC) and are engineered to express increasing quantities of humanthymidylate synthase (Hu TS) from the cloned cDNA. Transfection is donein a transient or permanent basis (see Chen, L. et al. (1996), HudziakR. M. et al. (1988), and Carter, P. et al. (1992). NIH-000(ras-transformed parent cell line); NIH-001 (low expresser of HuTS);NIH-002 (intermediate expressor of Hu TS); NIH-003 (high expressor ofHuTS). The level of expression of TS in each cell line is monitored byimmunoblot and enzyme assay in cell substrates, using antibody directedversus HuTS protein for immunodetection (e.g., as described in Chen, L.et al. (1996)). Enzymatic assays are performed as reviewed by Carreras,C. W. and Santi, D. N. (1995).

Human colorectal and breast tumor cell lines are screened for expressionof HuTS enzyme. Cell lines expressing low, moderate and high levels ofHuTS will be exposed to drug candidates as described above for the NIH3T3 cell lines. Growth inhibition and cytotoxicity are monitored asdescribed above. Similar tests can be carried out for each of theenzymes listed in Table 1.

In Vivo Testing

Ras-transformed NIH 3T3 cell lines are transplanted subcutaneously intoimmudeficient mice. Initial therapy may be direct intratumoralinjection. The expected result is that increased level of expression ofHuTS or a target enzyme leads to enhanced antitumor activity by the drugcandidates. Similar studies are performed with human tumors expressingincreasing levels of HuTS or a target enzyme, and demonstrating Nefficacy in response to drug correlates with their level of HuTSexpression or target enzyme. Optionally, experiments are be performed asabove except the drug will be administered intravenously into theanimals to address issues related to efficacy, toxicity andpharmacobiology of the drug candidates.

The in vivo studies will be conducted as described by Harris, M P et al.(1996) and Antelman, D. et al. (1995).

While the invention has been described in detail herein and withreference to specific embodiments thereof, it will be apparent to oneskilled in the art that various changes and modifications can be made tothe invention as described above without departing from spirit and scopeof.

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1-36. (canceled)
 37. A method for identifying potential therapeutic agents, comprising: contacting a target cell with a candidate therapeutic agent that is a selective substrate for a target enzyme, under conditions that favor the incorporation of the agent into the intracellular compartment of the target cell; and assaying the target cell for inhibition of cellular proliferation or cell killing.
 38. A method for selecting a uridine analogue for reducing or inhibiting the replication or spread of tumor cells, comprising the steps of: providing a uridine analog unsubstituted in the 5-position; testing said uridine analog for activation by thymidylate synthase; and selecting said uridine analog when found to be activated by thymidylate synthase.
 39. The method according to claim 38, wherein the testing step comprises: measuring cytotoxicity of said uridine analogue with respect to at least one cell line with a high expression of thymidylate synthase enzyme and at least one cell line with a low expression of thymidylate synthase enzyme; and the selecting step comprises: selecting said uridine analogue when the cytotoxicity measured with respect to the at least one cell line with a high expression of thymidylate synthase enzyme is greater than the cytotoxicity measured with respect to the at least one cell line with a low expression of thymidylate synthase enzyme.
 40. The method according to claim 38, wherein the uridine analog contains a radioisotope.
 41. The method of claim 38 wherein the uridine analogue is a compound of the following general formula:

wherein: A=N, C; B=H, hydroxyl, halogen, acyl (C₁-C₆), alkyl (C₁-C₆), alkoxy (C₁-C₆); D=O, S, NH₂; and G=substituted or unsubstituted cyclic sugar, substituted or unsubstituted acyclic sugar, substituted or unsubstituted mono, di, or tri-phospho-cyclic-sugar phosphate; substituted or unsubstituted mono, di, or tri-phospho-acyclic-sugar phosphate; substituted or unsubstituted mono, di, or tri-phospho-cyclic sugar analogues; substituted or unsubstituted mono, di, or tri-phospho-acyclic sugar analogues wherein the substituents are alkyl (C₁-C₆), alkoxy (C₁-C₆) or halogen.
 42. The method according to claim 41, wherein the compound contains a radioisotope.
 43. A method of imaging an organism, comprising the steps of: contacting the organism to be imaged with a compound of the formula:

wherein: A=N, C; B=H, hydroxyl, halogen, acyl (C₁-C₆), alkyl, alkoxy (C₁-C₆); D=O, S,NH₂; E=H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, halogen, or any substituent which is readily cleaved in the body to generate H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, or halogen; at least one of W, X, Y, Z is a label or a label containing moiety having sufficient isotopic activity for imaging and the remainder of W, X, Y, Z=H, hydroxyl, halogen, alkyl (C₁-C₆), substituted alkyl (C₁-C₆), alkoxy (C₁-C₆), substituted alkoxy (C₁-C₆); J=C, S; and K=O, C; and imaging the organism.
 44. A method of determining the proliferation rate of a tissue, comprising the steps of: contacting the tissue with a compound of the formula:

wherein: A=N, C; B=H, hydroxyl, halogen, acyl (C₁-C₆), alkyl, alkoxy (C₁-C₆); D=O, S,NH₂; E=H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, halogen, or any substituent which is readily cleaved in the body to generate H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, or halogen; at least one of W, X, Y, Z is a label or a label containing moiety having sufficient isotopic activity for imaging and the remainder of W, X, Y, Z=H, hydroxyl, halogen, alkyl (Cl-C₆), substituted alkyl (Cl-C₆), alkoxy (Cl-C₆), substituted alkoxy (C₁-C₆); J=C, S; and K=O, C; imaging the tissue; and determining the amount of the compound incorporated into the tissue, wherein the amount of the compound incorporated into the tissue correlates to the proliferation rate of the tissue.
 45. The method of claim 44, wherein the compound is a labeled uridine analog.
 46. A method of assessing the response of tumors to treatment with a thymidylate synthase inhibitor, comprising the steps of: (a) administering a uridine analogue selected by a method comprising the steps of: providing a uridine analogue unsubstituted in the 5-position; testing said uridine analog for activation by thymidylate synthase; and selecting said uridine analog when found to be activated by thymidylate synthase; wherein said uridine analogue is labeled with a positron emitter; and (b) determining an extent of maximum thymidylate synthase inhibition and persistence of thymidylate synthase inhibition over time between doses by external imaging.
 47. A method for assessing the response of tumors to treatment with a combination of thymidylate synthase inhibitor and at least one additional compound with antitumor activity, comprising the steps of: (a) administering a combination of a uridine analogue and at least one additional compound with anti-tumor activity, wherein said uridine analogue is selected by a method comprising the steps of: providing a uridine analogue unsubstituted in the 5-position; testing said uridine analog for activation by thymidylate synthase; and selecting said uridine analog when found to be activated by thymidylate synthase; wherein said uridine analogue is labeled with a positron emitter; and (b) determining an extent of maximum thymidylate synthase inhibition and persistence of thymidylate synthase inhibition over time between doses by external imaging.
 48. The method of claim 47, wherein said at least one additional compound with anti- tumor activity comprises a uridine analog.
 49. A compound of the formula:

wherein: A=N, C; B=H, hydroxyl, halogen, acyl (C₁-C₆), alkyl, alkoxy (C₁-C₆); D=O, S, NH₂; E=H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, halogen, or any substituent which is readily cleaved in the body to generate H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, or halogen; at least one of W, X, Y, Z is a label or a label containing moiety having sufficient isotopic activity for imaging and the remainder of W, X, Y, Z=H, hydroxyl, halogen, alkyl (Cl-C₆), substituted alkyl (Cl-C₆), alkoxy (Cl-C₆), substituted alkoxy (C₁-C₆); J=C, S; and K=O, C.
 50. A compound according to claim 49, wherein E is selected from H, methyl and iodine.
 51. A method of diagnosing tumors which are resistant to thymidylate synthase inhibitors, comprising the steps of: (a) administering a uridine analogue selected by a method comprising providing a uridine analogue unsubstituted in the 5-position; testing said uridine analog for activation by thymidylate synthase; and selecting said uridine analog when found to be activated by thymidylate synthase prior to obtaining biopsy specimens; (b) obtaining biopsy specimens; and (c) analyzing DNA from the biopsy specimens for extent of analogue incorporation.
 52. A method of diagnosing tumors which are resistant to thymidylate synthase inhibitors, comprising the steps of: (a) administering to said tumors a uridine analogue labeled with a positron emitting isotope selected by a method comprising providing a uridine analogue unsubstituted in the 5-position; testing said uridine analog for activation by thymidylate synthase; and selecting said uridine analog when found to be activated by thymidylate synthase; and (b) analyzing DNA incorporation in said tumor by external imaging.
 53. A method of treating cancer, comprising: administering a uridine analog in an amount effective to inhibit the replication or spread of cancer cells, said uridine analog selected by a method comprising the steps of: providing a uridine analogue unsubstituted in the 5-position; testing said uridine analog for activation by thymidylate synthase; and selecting said uridine analog when found to be activated by thymidylate synthase. 